The invention relates to methods and materials for controlling insect species. In particular, the invention relates to insecticidal proteins, DNA sequences encoding such proteins, and the genetic manipulation of plants and other organisms.
Numerous insect species are serious pests to common agricultural crops such as corn, soybeans, peas and similar crops. During the last century, the primary method of controlling such pests has been through the application of synthetic chemical insecticidal compounds. However, as the use of such chemical compounds proliferated and continued, it became evident that such wide-spread use posed problems with regard to the non-selectivity of the compounds, the increasing insect resistance to the chemicals and the environmental affect of such compounds, after run-off, on higher order species such as fish and birds among others. As a result of such problems, other methods of controlling insect pests were sought and tried.
One such alternative method of pest control has been the use of biological organisms which are typically xe2x80x9cnatural predatorsxe2x80x9d of the species sought to be controlled. Such predators may include other insects, fungi (milky-spore) and bacteria such as Bacillus thuringiensis cv. Alternatively, large colonies of an insect pest have been captively raised, sterilized and released into the environment in the hope that mating between the sterilized insects and fecund wild insects will decrease the insect population. While both these approaches have had some success, they entail considerable expense and present several major difficulties. For example, it is difficult both to apply living biological organisms to large areas and to cause such organisms to remain in the treated area or on the treated plant species for an extended time. Predator insects can migrate and fungi or bacteria can be washed off a plant or removed from a treated area by rain. Consequently, while the use of such biological controls has desirable characteristics and has met with some success, in practice these methods seem severely limited. However, scientific advances seem to offer new opportunities for controlling insect pests.
Advances in biotechnology in the last two decades have presented new opportunities for pest control through genetic engineering. In particular, advances in plant genetics coupled with the identification of insect growth factors and naturally-occurring plant defensive compounds or agents offer the opportunity to create transgenic crop plants capable of producing such defensive agents and thereby protect the plants against insect attack.
Scientists have identified some specific plant components or compounds which act as defensive agents to protect a plant from attack by insect pests and pathogens. While such components are usually present at only low levels in various plant tissues, some of them are also capable of being induced to higher levels upon attack by an insect pest or a pathogen. Examples of such defensive compounds include alkaloids, terpenes, and various proteins such as enzymes, enzyme inhibitors, and lectins.
Of particular interest are enzyme inhibitors that can block enzymatic activity and inhibit insect growth. For example, trypsin is a digestive enzyme. Its role in a body is to hydrolyze polypeptides into smaller units which can then be utilized by the host subject, for example, an insect. Blocking trypsin activity will inhibit insect growth. A trypsin inhibitor (abbreviated TI) is thus a compound which will block or decrease trypsin protease activity. As a result of such blockage or decrease in trypsin protease activity, a host subject which has ingested TI with its food will obtain little or no benefit from the polypeptides contained in the food. The host may thus fail to grow, mature and may indeed ultimately starve and die.
Trypsin inhibitors and lectins have been reported in the seeds of a number of leguminous tropical plants. The proposed role of such trypsin inhibitors (TIs) in plant defense has been shown using transgenic plants expressing a TI gene. Hilder et al. (1987) Nature 330:160-163, introduced the Bowman-Birk TI gene from soybeans into tobacco plants and showed that the transgenic plants were able to resist damage from a lepidopteran insect. Transformation and expression of other TI genes such as potato TI I and II also resulted in transgenic plants which showed resistance to insect attack. In the same experiments, transgenic plants which contained an unexpressed TI gene were susceptible to insect attack (See, Johnson et al. (1989) Proc. Natl. Acad. Sci. USA 86:9871-9875).
Transgenic plants that are resistant to specific insect pests have been produced using genes encoding Bacillus thuringiensis (Bt) endotoxins or plant protease inhibitors (PIs). Transgenic plants containing Bt endotoxin genes have been shown to be effective for control of some insects. Effective plant protection using transgenically inserted PI genetic material has not yet been demonstrated in the field. While cultivars expressing Bt genes may presently exhibit resistance to some insect pests, resistance based on the expression of a single gene might eventually be lost due to the evolution of Bt resistance in the insects. Thus, the search for additional genes that can be inserted into plants to provide protection from insect pests are needed.
Compositions and methods for the control of insects and other pests are provided. The compositions comprise proteins having pesticidal activities which can be isolated from leguminous plants. Particularly, the invention identifies and provides trypsin inhibitors obtainable from the genus, Pentaclethra, said inhibitors being approximately 38-45 and approximately 6-9 kilodaltons (kDa) in size. Purified protein, as well as amino acid and DNA sequence information is provided for proteins having insecticidal activity. The DNA sequences encoding the pesticidal proteins can be used to transform plants, bacteria, and other organisms for the control of pests.
The compositions and methods of the invention may be used in a variety of systems for controlling plant and non-plant pests.